Experimental device designed for assay and purification of oxygen-sensitive enzymes and its application.

一个厌氧手套箱，配备了一个净化装置和分光光度计，作为一个试验制造和使用。丙酮酸甲酸裂解酶（PFL）在口腔链球菌无氧糖代谢中起重要作用。PFL对氧非常敏感，并假定在催化位点附近含有蛋白质自由基。分子氧与自由基的反应似乎产生进一步的有害活性氧，并迅速导致酶的不可逆失活。由于不含自由基，酶的可逆失活形式是耐氧的，可被PFL激活酶激活。部分纯化的PFL激活酶很不稳定，但用亚铁离子、硒离子、蝶呤孵育，可恢复部分活性。该酶似乎含有一种“蝶呤样辅因子”，这种辅因子被认为存在于黄嘌呤氧化酶中。变形链球菌的激活酶是由丙酮酸而不是草氨酸触发的，并且非常不稳定。链球菌酶的性质被认为与大肠杆菌酶的性质不同，在厌氧条件下，菌斑微生物保持胞内NADH/NAD平衡是必需的。口腔链球菌通过“PFL系统”维持平衡。乳酸杆菌将乙酸代谢为乙醇氧化还原NADH，放线菌在碳酸氢盐存在的条件下通过琥珀酸途径再生NAD，提高产酸速率。

An anaerobic glove box, equipped with a purification apparatus and a spectorophotometer, was manufactured as a trial and employed. Several interesting finding were turned out as follows.Pyruvate formate-lyase (PFL) plays an important role in anaerobic sugar metabolism in oral streptococci. PFL was extremely sensitive to oxygen, and assumed to contain a protein radical near a catalytic site. The reaction of molecular oxygen with the radical seemed to generate further harmful active oxygen and bring about irreversible inactivation of the enzyme rapidly. Reversible inactive form of the enzyme was oxygen torelant since it did not contain the radical, and could be activated by PFL-activating enzyme. Partially purified PFL activating enzyme was quite unstable, but incubation of the enzyme with ferrous ion, selenium ion, pterin, restored a part of the activity. The enzyme seemed to contain "a pterin like cofactor" which was assumed to be exist in xanthine oxidase. The activating enzyme of Streptococcus mutans was triggered by pyruvate, but not oxamate, and quite unstable. The properties of the streptococcal enzyme is considered different from that of Escherichia coli.It is essential for plaque microorganisms to keep intracellular NADH/NAD balance under anaerobic conditions. Oral streptococci employed "the PFL system" to keep the balance. Lactobacilli metabolized acetate into ethanol to oxidize NADH to keep it. Actinomyces could regenerate NAD via succinate pathway when bicarbonate was available, and increased the rate of acid production.